Many molecular biology techniques depend on cloning individual cells from a mixture of cells.
For example, in the production of monoclonal antibodies, an essential step is hybridoma selection, including the separation and culture of individual hybridoma clones (fused myelomas and primary mouse cells). After cell fusion, the traditional way of selecting for monoclonality is to plate out single cells into 96-well dishes. This is repeated until clonality is assured.
Similarly, understanding gene function and identification of pharmaceutical leads requires the establishment of cell lines containing transfected genes expressed at an appropriate level. Standard techniques require the co-transfection of a gene with a dominant selectable marker followed by selection for growth for example in an antibiotic such as G418 or hygromycin. The resulting colonies are then picked by hand and further analysed for gene expression (RT-PCR) and functional expression.
Ascertaining optimal conditions for cell growth and differentiation requires broad testing of growth factors and culture conditions. The evaluation of a particular treatment requires a statistical approach on a large number of individual cells. One way to achieve this is to use numerous culture dishes, several for each treatment.
This process of cloning out may be modified and automated through the use of robots. Thus, for example, the ClonePix robot (manufactured by Genetix) implements this process by picking individual colonies directly from standard semi-solid media, the media preventing migration of the dividing cells. Thus, an imaging head captures images of colonies growing in the medium under white light, and software routines allow the separation and detection of individual colonies. A picking head then picks individual colonies into a 96-well plate.
Using a robot implemented picking method, colonies can be picked into 96-well plates at a picking speed of up to 400 clones per hour and graphic software allows the user to select colonies on the basis of size, shape, brightness and proximity. Furthermore, the software allows stratification of clones into slow, medium and fast growing cells, and clones of the same class may be grouped in the same 96-well plate. This gives rise to considerable savings in subsequent tissue culture steps as all wells can be processed at the same time.
When selecting cell colonies, it is desirable to identify those which are most productive for the polypeptide of interest. The robot implemented cloning method may identify and select cell colonies based on visualisation of colony size. Thus, the image capture system provides information on the size of the colony, and all colonies within a certain size range are picked. Effectively this system assumes that productivity of the desired polypeptide is directly related to size of the colony.
However this assumption is not necessarily correct. It is known for example that different hybridoma clones are capable of producing varying amounts of antibody, regardless of colony size. When selecting colonies based purely on colony size, no direct information is provided or processed as to the productivity of different cells (i.e., the quantity of product produced or secreted). Thus this system cannot discriminate between a high-producing hybridoma cell or colony and a low-producing hybridoma cell or colony if they are of a similar size. With regard to transfected cells, the robot cannot distinguish between clones with different levels of expression and/or secretion of recombinant protein.
A method disclosed in EP1752771 addresses this issue by identifying cells producing a polypeptide of interest using a combination of a class marker and a specificity marker. Marker-polypeptide complexes can then be detected, for example by an automated imaging system, and cells producing a high level of the polypeptide picked directly by a robot.
Cell or colonies producing a polypeptide of interest can also be selected by the use of dominant selectable marker. A gene for the marker may be transfected into cells together with a gene for the polypeptide of interest, followed by amplification of the marker gene. The marker is typically an enzyme, and amplification of the marker gene may occur in increasing concentrations of an inhibitor of the enzyme. The most commonly used system for such co-amplification uses dihydrofolate reductase (DHFR) as the enzyme. DHFR can be inhibited by the drug methotrexate (MTX). To achieve co-amplification, a host cell (which may lack an active gene which encodes DHFR) is transfected with a vector which comprises DNA sequences encoding DHFR and a desired protein. The genes for DHFR and desired protein may also be co-transfected into the cell on different vectors. The transfected host cells are cultured in media containing increasing levels of MTX, and those cell lines which survive are selected.
In a further method, productive colonies may be identified by binding a marker to a reference polypeptide whose expression is linked to the polypeptide of interest. For instance, the reference polypeptide may be an amplifiable selectable marker such as DHFR and the marker may be a labelled molecule which binds to the marker (e.g. methotrexate for DHFR). The cell colonies may be imaged to identify labelled cells in which the marker is amplified. These colonies are also expected to produce the polypeptide of interest at a high level.
In some of the above methods, productive colonies may be identified by imaging of a signal associated with the polypeptide of interest. The signal may be derived from a marker (e.g. an antibody or inhibitor) which binds directly to the polypeptide of interest, or which binds to another molecular species which is indicative of the level of the polypeptide of interest in the cell. Typically the signal is a fluorescent signal (e.g. from a fluorescently-labelled antibody or inhibitor).
However, in these methods there is still a problem of how to correlate the signal associated with the marker with the most productive colonies. In particular, it is not apparent how image data should be interpreted in order to allocate signal values to a particular colony in the most efficient manner. Moreover, it is not clear whether the colonies which produce the polypeptide of interest at the highest level are simply those which are associated with the highest signal.
Therefore there is still a need for an improved method for identifying a cell or cell colony, which allows the most productive cells or colonies to be identified from image data of the type discussed above.